Construction of a new polycistronic vector for over-expression and rapid purification of human hemoglobin.
To facilitate the study of the structure-function relationship of human hemoglobin (Hb A), we have developed a new hemoglobin expression vector, pGEX6P-alpha-[SD]-beta. This vector allows the co-expression of alpha-Hb as a fusion protein with Glutathione S-Transferase (GST-alpha-Hb) and beta-Hb with an additional methionine at the N-terminal extremity (rbeta-Hb). These proteins were solubilized as GST-alpha-Hb/rbeta-Hb complex form and purified in one step by affinity chromatography on immobilized glutathione. The CO binding kinetic studies show that the GST-alpha-Hb/rbeta-Hb complex and recombinant Hb A exhibit the same allosteric behavior as for native Hb A. The GST moiety, which does not modify the function of the complex, can be easily eliminated by cleavage by the PreScission Protease. After cleavage during the rapid purification procedure, over 20mg of recombinant Hb per liter of culture were obtained, more than double the yield of previous co-expression systems. This polycistronic vector system, which offers the additional advantage of a very rapid purification, is especially well suited for the study of abnormal, unstable globins in order to better understand the associated pathology.
Domingues
Elisa
E
INSERM U779, University of Paris VII and XI, Le Kremlin-Bicêtre, France.
Brillet
Thomas
T
Vasseur
Corinne
C
Agier
Virginie
V
Marden
Michael C
MC
Baudin-Creuza
Véronique
V
D002248:Carbon Monoxide / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D015870:Gene Expression; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006441:Hemoglobin A / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D055542:alpha-Globins / Q000235:genetics; D055544:beta-Globins / Q000235:genetics
D011993:Recombinant Fusion Proteins; D055542:alpha-Globins; D055544:beta-Globins; D002248:Carbon Monoxide; D006441:Hemoglobin A; D005982:Glutathione Transferase